Slides were placed in QAR cassettes (FujiFilm Life Sciences, Stamford, CT) along with125I autoradiographic standards (Amersham Biosciences). A phosphor screen (FujiFilm Life Sciences, 20×40 super-resolution) was placed on the slides and standards and allowed to develop for up to 14days. QAR phosphor screens were developed in a high-resolution phosphor-imager (FUJI FLA-7000, FujiFilm Life Sciences) and converted to digital images. Digital QAR images were calibrated to125I standards and analyzed using MCID Analysis software (InterFocus Imaging LTD, Linton, Cambridge, England). Metastases permeability fold-changes were calculated based on125I signal intensity within confirmed metastases locations (determined by eGFP fluorescence image overlays) relative to125I signal intensity in normal brain.
Fuji fla 7000
Manufactured by Fujifilm
The FUJI FLA-7000 is a versatile lab equipment designed for fluorescence detection and imaging. It features a high-performance CCD camera and advanced optics to capture high-quality images of fluorescent samples.
Automatically generated - may contain errors
Lab products found in correlation
Qar cassettes, by Fujifilm (2 mentions)
Quantitative autoradiography cassettes, by GE Healthcare (2 mentions)
Sodium deoxycholate, by MP Biomedicals (1 mentions)
Coomassie plus protein assay, by Thermo Fisher Scientific (1 mentions)
1 2 dipalmitoyl sn glycerol 3 phosphocholine, by Avanti Polar Lipids (1 mentions)
Cellsens image analysis software, by Olympus (1 mentions)
Mvx10 stereomicroscope, by Olympus (1 mentions)
Orca flash4.0 v2 scmos, by Hamamatsu Photonics (1 mentions)
Cm3050 cryostat, by Leica (1 mentions)
Mvx10, by Olympus (1 mentions)
Immobilon ny membrane, by Merck Group (1 mentions)
6 protocols using fuji fla 7000
1
Quantifying Metastatic Tumor Permeability
2
Quantitative Phospholipase D Activity Assay
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Protein concentration of the soluble fraction was determined using a Coomassie Plus Protein Assay (Thermo Scientific). To measure PLDα activity, 15 µg protein were used per assay. The substrate solution consisted of 8 µM fluorescent PC (BODIPY-PC, D3792, Life Technologies), 50 µM 1,2-dipalmitoyl-sn-glycerol-3phosphocholine (Avanti Polar Lipids), 0.015% sodium deoxycholate (MP Biomedicals), and 50 mM MES buffer (pH 6.5). The substrate solution was shaken gently at 23°C for 30 min, followed by sonication for 10 min. The reaction solution contained 20 mM CaCl 2 , 0.4% n -butanol (Lachema), and 25 μL of substrate solution. The reaction was initiated by adding protein solution, and incubated at 28°C with 300 rpm for 30 min. Lipids were extracted and run through HP-TLC silica gel-60 plates as in Krčkováet al. (2018) . Plates were developed in a mobile phase of acetone/ethanol (1/1, by vol.) and laser-scanned using a Fuji FLA-7000 fluorescence scanner. Phosphatidylbutanol spots were quantified using the Fuji FLA-7000 quantification software Multigauge (version (Fujifilm, Japan). BODIPY-phosphatidylbutanol was identified after comparison to the standard, prepared using commercially available PLD (Sigma) (Pejcharet al. , 2010) .
3
Multimodal Fluorescent and Autoradiographic Analysis
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Fluorescent analyses were performed using an Olympus MVX10 stereomicroscope (Olympus, Tokyo, Japan) (optical zoom range 0.63–12.6, NA = 0.5) with a Hamamatsu ORCA Flash4.0 v2 sCMOS and DAPI/FITC/RFP/Cy5/Cy7 filter set. Sections were imaged using RFP (588 nm) channel to detect 3 kDa Texas red (TxRd) dextran. CellSens image analysis software (Olympus, Tokyo, Japan) was used to quantitate 3 kDa TxRd dextran accumulation. The same slides were placed in quantitative autoradiography cassettes (GE Healthcare Life Sciences, Chicago, IL) with corresponding 14C standards (0.1–862 nCi/g). A 20 × 40 super-resolution phosphor screen (Fujifilm Life Sciences, Cambridge, MA) was placed over the slides and developed for 21 days. Screens were read on FUJI FLA-7000 (Fujifilm Life Sciences, Cambridge, MA) high-resolution phosphor imager. Quantification of 14C-AIB was analyzed with MCID Analysis Software (InterFocus Imaging, Cambridge, England). Accumulation of 14C-AIB is reported as nCi/g while 3 kDa TxRd accumulation is sum intensity/area.
4
Multimodal Brain Imaging and Analysis
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Following tracer circulation, brains were harvested, flash-frozen in isopentane at − 70 °C. Sectioning was performed using a Leica CM3050 cryostat (Leica Microsystems, Los Angeles, CA) to obtain 20 µm thick slices. Data were analyzed using an Olympus MVX10 fluorescent, stereomicroscope (optical zoom range 0.63–12.6, N.A = 0.5). Sections were imaged using RFP (588 nm), and DAPI (461 nm) channels to detect Texas red (TxRed) and cascade blue (10 kDa dextran) respectively. The same sections were placed in quantitative autoradiography cassettes (GE Healthcare) with corresponding 14C standards (0.1–862 nCi/g). A phosphor screen was placed over the samples (Fujifilm Life Sciences, 20 × 40 super-resolution) and slides were allowed to develop for 21 days. Screens were read on a high-resolution phosphor imager (FUJI FLA-7000, Fujifilm, Life Sciences). Permeability of 14C-AIB was analyzed using MCID Analysis (InterFocus Imaging LTD). The same slides were then stained with 0.1% Cresyl violet and imaged for histopathological visualization.
5
tRNA and RNA Separation and Detection
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0.4 μg of bulk tRNA or 1 μg of total RNA per sample were heated for 5 min at 80 °C and applied onto denaturing polyacrylamide gels (8 % polyacrylamide (19:1), 7 M urea, with or without 0.05 % ([N-acryloylamino]phenyl)mercuric chloride (APM) in 0.5 × TBE) (31 (link)). RNA was transferred onto Immobilon NY+ membranes (Millipore) by semi-dry transfer with 0.5× TBE. Northern blot was performed essentially as described using specific DNA-oligo probes (21 (link)). Signals were collected and analysed using a Fuji FLA 7000 phosphoimager (Fujifilm). Digital image analysis was performed using Fiji-ImageJ2 (The Fiji Project).
6
Quantitative Autoradiography of Metastatic Brain
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